Biochemical Characterization of MiddleEast Respiratory Syndrome CoronavirusHelicase

Adeyemi O. Adedeji, Hilary LazarusDepartment of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University,Glendale, Arizona, USA

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is asuperfamily 1 helicase containing seven conserved motifs. We have cloned, ex-pressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13(M-nsp13) helicase. Characterization of its biochemical properties showed that it un-wound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5=-to-3= direction and effi-ciently unwound the partially duplex RNA substrates with a long loading strandrelative to those of the RNA substrates with a short or no loading strand. Moreover,the Km of ATP for M-nsp13 is inversely proportional to the length of the 5= loadingstrand of the partially duplex RNA substrates. Finally, we also showed that the rateof unwinding (ku) of M-nsp13 is directly proportional to the length of the 5= loadingstrand of the partially duplex RNA substrate. These results provide insights that en-hance our understanding of the biochemical properties of M-nsp13.

IMPORTANCE Coronaviruses are known to cause a wide range of diseases in hu-mans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is anovel coronavirus discovered in 2012 and is responsible for acute respiratory syn-drome in humans in the Middle East, Europe, North Africa, and the United States ofAmerica. Helicases are motor proteins that catalyze the processive separation ofdouble-stranded nucleic acids into two single-stranded nucleic acids by utilizing theenergy derived from ATP hydrolysis. MERS-CoV helicase is one of the most impor-tant viral replication enzymes of this coronavirus. Herein, we report the first bacterialexpression, enzyme purification, and biochemical characterization of MERS-CoV heli-case. The knowledge obtained from this study might be used to identify an inhibitorof MERS-CoV replication, and the helicase might be used as a therapeutic target.

KEYWORDS: ATP hydrolysis, DNA, RNA, coronavirus, enzyme kinetics, helicase

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirusdiscovered in 2012 and is responsible for acute respiratory syndrome in humans

in the Middle East (Saudi Arabia, Jordan, Qatar, and the United Arab Emirates), Europe(the United Kingdom, France, Italy, and Germany), North Africa (Tunisia and Egypt), andthe United States of America (1, 2). Globally, as of 16 May 2016, the WHO has beennotified of 1,733 laboratory-confirmed cases of infection with MERS-CoV, including atleast 628 related deaths, in 27 different countries (http://www.who.int/csr/don/16-may-2016-mers-saudi-arabia/en/). While gastrointestinal symptoms, including diarrhea andqueasiness, are also occasionally observed (3, 4), MERS-CoV causes mainly respiratorydiseases.

MERS-CoV is a positive single-stranded RNA (ssRNA) virus with one of the largestknown RNA genomes (30.119 kb) (5). Following infection, there is translation of twolarge replicative polyproteins, pp1a (the ORF1a polyprotein) and pp1ab (the polypro-

Received 12 August 2016 Accepted 19August 2016 Published 7 September 2016

Citation Adedeji AO, Lazarus H. 2016.Biochemical characterization of Middle Eastrespiratory syndrome coronavirus helicase.mSphere 1(5):e00235-16.doi:10.1128/mSphere.00235-16.

Editor Matthew B. Frieman, University ofMaryland

Copyright © 2016 Adedeji and Lazarus. This isan open-access article distributed under theterms of the Creative Commons Attribution 4.0International license.

Address correspondence to Adeyemi O.Adedeji, aadede@midwestern.edu.

RESEARCH ARTICLEMolecular Biology and Physiology

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tein made from ORF1a and ORF1b through a �1 ribosomal frameshift during transla-tion). These polyproteins are processed by the virus-encoded papain-like proteinase(PLpro) and nonstructural protein 5 (nsp5), a 3C-like protease (3CLpro). This auto-proteolysis leads to the formation of 16 nonstructural proteins, including an RNA-dependent RNA polymerase (RdRp) and a nucleoside triphosphatase (NTPase) andhelicase that are known as nsp12 and nsp13, respectively (3, 6–8). These are likely toform the core of membrane-bound replication-transcription complexes in double-membrane vesicles at perinuclear regions (9).

Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids (ds) into two single-stranded nucleic acids (ss) by utilizing theenergy derived from ATP hydrolysis (10–17). Helicases are known to unwind nucleicacids during replication, recombination, and DNA repair (14), and recent reports haveshown that they are also involved in other biological processes, including displacementof proteins from nucleic acid, movement of Holliday junctions, chromatin remodeling,catalysis of nucleic acid conformational changes (18–23), and several aspects of RNAmetabolism (transcription, mRNA splicing, mRNA export, translation, RNA stability) andmitochondrial gene expression (9). A more recent biochemical study revealed a dualfunction of human enterovirus nonstructural protein 2CATPase as both an RNA helicaseand an ATP-independent RNA chaperone which may destabilize RNA duplexes andassist in the formation of more globally stable RNA structures (24). Defects in helicasefunction have been associated with some human diseases, including Bloom’s syn-drome, Werner’s syndrome, and xeroderma pigmentosum (25–28).

Previously, biochemical characterization of severe acute respiratory syndrome CoV(SARS-CoV) helicase (S-nsp13) demonstrated that S-nsp13 can unwind both double-stranded DNA and RNA in a 5=-to-3= direction, and it can hydrolyze all deoxyribonu-cleotide triphosphates (dNTPs) and ribonucleotide triphosphates (29, 30), Additionalstudy also showed that nsp12, the SARS-CoV RdRp, enhances the catalytic efficiency ofS-nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwindingby 2-fold (31). However, there are no reports on the biochemical characterization ofMERS-CoV helicase (M-nsp13).

Herein, we report the first bacterial expression, enzyme purification, and biochem-ical characterization of M-nsp13. With double-stranded RNA (dsRNA) as a substrate,M-nsp13 unwound the substrate in a 5=-to-3= direction, with the helicase requiringmore ATP for optimal unwinding of RNA substrates with short 5= loading strands. Wealso report that the rate of unwinding (ku) of M-nsp13 is directly proportional to thelength of the 5= loading strand of the partially duplex RNA substrate. While M-nsp13required a single-strand 5- to 20-nucleotide (nt) 5= overhang for efficient unwinding ofthe dsRNA substrate, M-nsp13 was still able to partially unwind (~10% to 30%) a dsRNAsubstrate with a 0-nt (blunt-ended) to 2-nt 5= loading strand.

RESULTSExpression and purification of MERS-CoV helicase (M-nsp13). To obtain sufficientamounts of M-nsp13 for biochemical studies, an Escherichia coli expression system wasused. MERS-CoV pp1ab residues 5311 to 5881 were fused at the N terminus to the E. coliStrep tag. As Fig. 1A shows (lanes E1 to E4), fusion protein Strep–M-nsp13 of sufficientpurity was obtained with a two-step purification protocol involving Strep-Tactin affinityand size exclusion chromatography. The same method was used to express and purifya Strep–M-nsp13 control protein, M-nsp13_K288A, in which the conserved lysineresidue (5598 in pp1ab) of the Walker A box (32) (helicase motif I in Fig. 2) was replacedwith Ala. The identities of the proteins were confirmed by Western blotting (Fig. 1B).

The amino acid sequence of M-nsp13 was aligned with the helicase amino acidsequences of other coronaviruses, including SARS-CoV isolate Frankfurt 1 (GenBankaccession no. AY291315), mouse hepatitis virus (MHV) strain A59 (accession numberNC_001846), porcine transmissible gastroenteritis virus (TGEV) strain Purdue 46 (acces-sion number AJ271965), and avian infectious bronchitis virus (IBV) strain Beaudette(accession number M95169). The protein alignment shows that all helicases, along with

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M-nsp13, have the conserved helicase motifs I to VI, with motif I (Walker A box)containing the Lys288 that is recognized as the NTP binding site and the core ATPasesite of superfamily 1 to 3 (SF1 to -3) helicases (32).

M-nsp13 can unwind both RNA and DNA helices. To evaluate the RNA helixunwinding activity of M-nsp13, various concentrations of the enzymes (1 to 40 nM)were incubated with a 5 nM concentration of the partially duplex RNA substrate witha 20-nt overhang (5=-RNA-20 [20 ss, 22 ds]) for 30 min. Unless stated otherwise, for thisspecific experiment, we used the 5=-RNA-20 partially duplex substrate (see Fig. S1 in thesupplemental material), since previous coronavirus helicase, i.e., SARS-CoV nsp13, wasshown to have a 5=-to-3= directionality (29). As shown in Fig. 3A, the substrate wascompletely unwound at a 20 nM enzyme concentration in a 5=-to-3= direction.

Some reports have shown that some helicases have a clear preference for either RNAor DNA (9, 33–36). To determine whether M-nsp13 prefers one substrate over the other,we used the 5=-overhang 20-nt (20 ss, 22 ds) RNA and DNA substrates (Fig. S1) andmonitored M-nsp13’s helicase activity. As shown in Fig. 3B, M-nsp13 did not exhibit anypreference for either RNA or DNA substrates. The results are similar to what waspreviously reported for SARS-CoV helicase (29). As such, our experimental analyses werecarried out using RNA substrates to mimic the natural substrate.

Metal and pH dependence of M-nsp13. To determine the metal requirementsof M-nsp13 for its helicase activity, a 20 nM enzyme concentration was incubated withthe 5=-RNA-20 (20 ss, 22 ds) substrate in the presence of 2 mM MnCl2, MgCl2, ZnCl2, or

FIG 1 Protein expression and purification. (A) M-nsp13 and FMDV polymerase (pol) genes werecloned in pET52b and pet-28b, respectively, followed by their expression in the E. coli BL21 bacterialexpression system. M-nsp13 was then purified by Strep-Tactin affinity chromatography. The FMDVpolymerase was purified using nickel affinity chromatography. Purified M-nsp13 and FMDV poly-merase were readily visualized by Coomassie blue staining of SDS-PAGE gels as ~68-kDa and 55-kDaprotein products, respectively, in line with their expected molecular masses. (B) Western immunoblotanalysis with M-nsp13-specific rabbit antiserum. The positions of protein molecular mass markers areindicated on the left (in kilodaltons).

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FIG 2 Sequence comparison of coronavirus helicases. The alignment was generated with theUniProt program (http://www.uniprot.org/align/) and the nsp13 sequences of MERS-CoV (humanbetacoronavirus strain 2c EMC/2012; GenBank accession no. JX869059.2), SARS-CoV (isolate Frankfurt

(Continued)

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CaCl2. The results showed that M-nsp13 had the best helicase activity with MgCl2,followed by MnCl2, with more than 85% of the substrate unwound. The enzymeshowed lesser activity with ZnCl2 and no observable activity in the presence of CaCl2(Fig. 4A).

To determine the optimal concentration of MgCl2 required for M-nsp13 activity,various concentrations of MgCl2 (1 to 6 mM) were incubated with 20 nM M-nsp13 andthe 5 nM 5=-RNA-20 (20 ss, 22 ds) substrate for 30 min. The result showed that M-nsp13had optimal activity with 2 mM MgCl2 (Fig. 4B).

To evaluate the optimal pH for M-nsp13 unwinding activity, reactions similar tothose described above were performed at various pHs (4.0 to 9.0). As shown in Fig. 5,M-nsp13 has optimal unwinding activity from pHs 7.0 to 8.5.

Figure Legend Continued1; accession no. AY291315), mouse hepatitis virus (MHV; strain A59; accession number NC_001846),transmissible gastroenteritis virus (TGEV; strain Purdue 46; accession number AJ271965), and avianinfectious bronchitis virus (IBV; strain Beaudette; accession number M95169) were derived from thereplicative polyproteins of these viruses, whose sequences were obtained from the GenBank data-base. Conserved helicase motifs I to VI are indicated. Also indicated by an @ sign is the conservedLys288 residue (corresponding to Lys5598 in pp1ab), which in the M-nsp13_K288A control proteinwas replaced with Ala. Lys288 is part of a conserved helicase motif (I) which is also called the WalkerA box. Near the N terminus, the 12 conserved Cys and His residues predicted to form a binuclearzinc-binding cluster are indicated by a circled “C.” Overall, MERS helicase has 72.4% identity withSARS, 67.2% identity with MHV, 61.3% identity with TGEV, and 59.1% with IBV helicases.

FIG 3 Helicase activity of M-nsp13. (A) Determination of optimal enzyme concentration. The activityof purified M-nsp13 was determined in an enzyme-dependent manner using a 5=-Cy3-labeled (*)partially duplex 5=-RNA-20 (20 ss, 22 ds) RNA substrate for 30 min. The reaction products wereseparated on a nondenaturing 8% polyacrylamide gel and visualized using the Bio-Rad multipurposeimager. (B) Comparison of fractions of partially duplex RNA and DNA substrates unwound byM-nsp13. The activity of purified M-nsp13 was verified in a time-dependent manner using a5=-Cy3-labeled (*) partially duplex 5=-RNA-20 (20 ss, 22 ds) RNA substrate (upper gel) and a 5=-DNA-20(20 ss, 22 ds) DNA substrate (lower gel). The reaction products were separated on a nondenaturing8% polyacrylamide gel and visualized using the Bio-Rad multipurpose imager.

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Minimum 5= loading strand length requirement of M-nsp13 for efficientRNA unwinding. To determine whether nucleic acid unwinding by M-nsp13 dependson the loading strand length and to determine the minimum length of the 5= single-stranded overhang required for efficient helicase activity, we designed five partiallyduplex RNA substrates with single-stranded 5= ends of various lengths (between 0 and20 bases) (see Fig. S1 in the supplemental material; Fig. 6) but with dsRNA duplexregions of the same length. Using these substrates, we monitored the unwinding

FIG 4 Determination of metal requirement. (A) The activity of purified M-nsp13 was performedusing a 5=-Cy3-labeled (*) partially duplex 5=-RNA-20 (20 ss, 22 ds) RNA substrate in the presence of2 mM of Zn2�, Ca2�, Mg2�, and Mn2�. The reaction products were separated on a nondenaturing 8%polyacrylamide gel and visualized using the Bio-Rad multipurpose imager. (B) Purified M-nsp13 wasincubated with the 5=-Cy3-labeled (*) partially duplex 5=-RNA-20 (20 ss, 22 ds) RNA substrate in thepresence of various concentrations of Mg. The reaction products were separated on a nondenaturing8% polyacrylamide gel and visualized using the Bio-Rad multipurpose imager. hel, helicase.

FIG 5 pH dependence. Purified M-nsp13 was incubated with the 5=-Cy3-labeled (*) partially duplex5=-RNA-20 (20 ss, 22 ds) RNA substrate in reaction buffers with different pHs (4.0 to 9.0). The reactionproducts were separated on a nondenaturing 8% polyacrylamide gel and visualized using theBio-Rad multipurpose imager.

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activity of M-nsp13 in the presence of 2 mM ATP. As shown in Fig. 6A, the efficiency ofRNA unwinding by M-nsp13 increased as the length of the 5= loading strand increased.While M-nsp13 required a 5- to 20-nucleotide (nt) single-strand 5= overhang for efficientunwinding of the dsRNA substrate, M-nsp13 was still able to partially unwind (~10% to30%) a dsRNA substrate with a 0- (blunt) to 2-nt 5= loading strand.

The optimal helix unwinding activity of M-nsp13 requires NTPs. NTP hydro-lysis is known to provide the energy required for translocation of RNA helicases alongssRNA and duplex-RNA during unwinding. To determine whether ATP is required forthe unwinding activities of M-nsp13, a standard unwinding assay was conducted withall the RNA substrates described above in the absence of ATP. As shown in Fig. 6B, whileM-nsp13 was partially able to unwind the partially duplex RNA substrate with a 20-ntoverhang (5=-RNA-20) in the absence of ATP, M-nsp13 could not unwind other RNAsubstrates with shorter loading strands.

FIG 6 Substrate specificity, minimum overhang length, and ATP requirement for M-nsp13. (A and B).Five different substrates with 5=-overhang lengths varying from 0 to 20 nucleotides were designedto determine the minimum length of the loading strand required by M-nsp13 to efficiently unwindits substrate. The helicase activity of M-nsp13 (20 nM) was assessed on these substrates (5 nM each)at 30°C with 2 mM ATP (A) and no ATP (B). The products were separated on a nondenaturing 8%polyacrylamide gel and visualized using the Bio-Rad multipurpose imager. (C) Purified M-nsp13 orM-nsp13_K288A (20 nM) was incubated with the 5=-Cy3-labeled (*) partially duplex 5=-RNA-20 (20 ss,22 ds) RNA substrate (5 nM) in the presence of 2 mM ATP. The reaction products were separated ona nondenaturing 8% polyacrylamide gel and visualized using the Bio-Rad multipurpose imager.

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A recently identified human enterovirus helicase, nonstructural protein 2CATPase, wasshown to possess an ATP-independent RNA chaperone activity (24). To investigatewhether M-nsp13 has an underlying ability to unwind its substrate in an ATP-independent manner, M-nsp13_K288A (the control protein that lacks the ability tohydrolyze ATP) was incubated with the 5=-RNA-20 partially duplex substrate in thepresence of 2 mM ATP. The results showed that M-nsp13_K288A could not unwind the5=-RNA-20 partially duplex substrate, indicating that M-nsp13 does not have an ATP-independent RNA chaperone activity (Fig. 6C). Overall, these results suggest thatM-nsp13’s ability to unwind the 5=-RNA-20 partially duplex substrate is likely due toresidual cellular ATP purified with the helicase as well as to a long overhang length,which enhances M-nsp13’s unwinding activity.

Given the observation that M-nsp13 was partially able to unwind the 5=-RNA-20partially duplex substrate when ATP was not included in the reaction mixture, wesought to investigate the ATP requirement of M-nsp13 for the different RNA substrateswith 5= loading strands of various lengths. We assessed the unwinding activity ofM-nsp13 with the various 5=-RNA partially duplex substrates with different overhanglengths in the presence of various ATP concentrations from 0.25 to 5 mM. Using theMichaelis-Menten equation, the unwinding activities under different ATP concentra-tions were plotted as the fraction of the released RNA from the total RNA helix substrateat each ATP concentration. The results showed that increasing ATP concentrations upto ~2 mM ATP apparently enhanced the helix unwinding of each RNA substrate(Fig. 7A), after which no significant difference in substrate unwinding was noticeable.Moreover, M-nsp13 could not completely unwind the 5=-RNA-0 (blunt substrate),5=-RNA-2 (2-nt overhang), and 5=-RNA-5 (5-nt overhang) partially duplex RNA substrateseven at 5 mM ATP. Furthermore, the Km and Vmax values for each substrate (Table 1)indicate that the amount of ATP required by M-nsp13 to unwind the substrates isinversely proportional to the length of the loading strand.

To further investigate the (d)NTP requirements of M-nsp13, a standard unwindingassay was conducted as described above, using the 5=-RNA-10 (10 ss, 22 ds [or 10-ntoverhang]) partially duplex RNA substrate in the presence or absence of individual(d)NTPs (2 mM). The results showed that M-nsp13 was able to hydrolyze all thenucleotides for the purpose of nucleic acid unwinding, with slightly more specificity forthe NTPs (Fig. 7B). These results further confirmed that the helix unwinding activity ofM-nsp13 needs the participation of ATP or other NTPs. It should be noted that sinceM-nsp13 can partially unwind the 5=-RNA-20 (20 ss, 22 ds [or 20-nt overhang]) partiallyduplex RNA substrate in the absence of ATP, we decided not to use this substrate forthis experiment to allow for proper assessment of the effect of NTPs on M-nsp13’sunwinding activity.

M-nsp13 rates of unwinding of the partially duplex RNA substrates thathave various 5= overhang lengths. To determine the rates of unwinding of M-nsp13with all the partially duplex RNA substrates with various 5= overhang lengths used inthis study, the enzyme was incubated with the RNA substrates 5=-RNA-0 (the bluntsubstrate), 5=-RNA-2 (2-nt overhang), 5=-RNA-5 (5-nt overhang), 5=-RNA-10 (10-nt over-hang), and 5=-RNA-20 (20-nt overhang) in the presence of 2 mM ATP at different timepoints (0 to 45 min). The change in the fractions of unwound ssRNA product with timeis shown in Fig. 8. The experimental data for the substrates were fit to the followingequation: fraction (ssDNA) � A · (1 – e– kt) � n, where A is the amplitude thatcorresponds to the maximum fraction of ssRNA that can be generated enzymaticallyfrom the substrates, k is the pseudo first-order rate constant of RNA unwinding, t is thereaction time, and n is an additive constant representing the amount of ssDNA presentbefore the reaction started. The results showed that the amplitudes and the rates ofRNA unwinding of M-nsp13 were directly proportional to the length of the 5= loadingstrand (Table 2).

M-nsp13 is a unidirectional helicase. Previous reports have shown that SARS-CoV helicase has a 5=-to-3= directionality and, as such, does not show any activity witha substrate that has a 3= overhang (29, 31). To determine whether M-nsp13 can also

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unwind in a 3=-to-5 direction, the standard helicase assay was conducted with the RNAsubstrates that have various lengths of the 3= loading strand in the presence of 2 mMATP. As shown in Fig. 9, M-nsp13 could not unwind any of the 3= RNA (3=-overhang)substrates, confirming that M-nsp13 is a unidirectional 5=-to-3= helicase.

DISCUSSION

Sequence alignment of M-nsp13 with other helicases suggests that the MERS-CoVhelicase belongs to the SF1 family of helicases (29). However, the biochemical aspects

FIG 7 (d)NTP requirements for MERS helicase-unwinding activities. (A) Five different 5=-Cy3-labeled(*) partially duplex RNA substrates with 5=-overhang lengths varying from 0 to 20 nucleotides, i.e.,0 ss, 22 ds (Œ), 2 ss, 22 ds (�), 5 ss, 22 ds (�), 10 ss, 22 ds ({), and 20 ss, 22 ds (�) RNA substrates,were reacted with M-nsp13 (20 nM) in the presence or absence of increasing concentrations (0 to5 mM) of ATP for 30 min. The reaction products were separated on a nondenaturing 8% polyacryl-amide gel and visualized using the Bio-Rad multipurpose imager. The unwinding activities underdifferent ATP concentrations were plotted as the fraction of the RNA released from the total RNAhelix substrate (y axis) at each ATP concentration (x axis) using the Michaelis-Menten equation. Thevalues for Km and Vmax are provided in Table 1. Error bars represent standard deviation (SD) valuesfrom two separate experiments. (B) The 5=-Cy3-labeled (*) partially duplex 5=-RNA-10 (10 ss, 22 ds)RNA substrate (5 nM) was reacted with M-nsp13 (20 nM) in the presence or absence of the indicatedNTPs (2 mM) for 30 min. The reaction products were separated on a nondenaturing 8% polyacryl-amide gel and visualized using the Bio-Rad multipurpose imager.

TABLE 1 Kinetic parameters for the ATP hydrolysis of M-nsp13 with partially duplex RNAsubstrates with loading strands of various lengthsa

RNA substrate Vmax (min�1) Km (ATP) (mM)

5=-RNA-0 (0 ss, 22 ds) 0.37 � 0.05 1.23 � 0.495=-RNA-2 (2 ss, 22 ds 0.47 � 0.04 1.14 � 0.285=-RNA-5 (5 ss, 22 ds) 0.72 � 0.03 0.62 � 0.095=-RNA-10 (10 ss, 22 ds) 0.99 � 0.02 0.41 � 0.045=-RNA-20 (20 ss, 22 ds) 0.99 � 0.02 0.26 � 0.03aThe Vmax and Km values were determined by fitting the data by nonlinear regression to the Michaelis-Menten equation using Prism 7.0 software. Experiments were performed two times.

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of the enzymatic mechanism of MERS-CoV nsp13, including ATP hydrolysis, transloca-tion along the nucleic acid, and the unwinding rate, have not been studied. The presentstudy fills this knowledge gap.

One of the unique findings of this study is the observation that the length of the 5=loading strand of the partially duplex RNA can determine the amount of ATP requiredby the helicase for optimal helix unwinding. The longer the length of the loadingstrand, the lower the amount of ATP required for optimal unwinding. As noted inFig. 6B, M-nsp13 was able to partially unwind the substrate with the longest 5= loadingstrand without inclusion of ATP in the reaction mixture. A recently characterized humanenterovirus helicase, nonstructural protein 2CATPase, was shown to possess an ATP-independent RNA chaperone activity (24). The RNA chaperone activity was observedalong the 3=-to-5= direction in the absence of ATP. Given the presence of an ssRNAproduct in the absence of ATP, when M-nsp13 was incubated with the 5=-RNA-20partially duplex RNA substrate, we wanted to confirm a possible RNA chaperone activityby M-nsp13. As shown in Fig. 6C, M-nsp13_K288A (the control protein that lacks theability to hydrolyze ATP) did not unwind either substrate, thereby ruling out an RNAchaperone activity by M-nsp13. The small amount of the ssRNA product observed withthe 5=-RNA-20 partially duplex RNA substrate in the absence of ATP may be due to

FIG 8 M-nsp13 rates of unwinding of all RNA substrates with 5= loading strands of various lengths.(A) Five different 5=-Cy3-labeled (*) partially duplex RNA substrates with 5=-overhang lengths varyingfrom 0 to 20 nucleotides, i.e., 0 ss, 22 ds (Œ), 2 ss, 22 ds (�), 5 ss, 22 ds (�), 10 ss, 22 ds ({), and 20 ss,22 ds (�), were reacted with M-nsp13 (20 nM) in the presence of 2 mM ATP for 0 to 45 min. Thereaction products were separated on a nondenaturing 8% polyacrylamide gel and visualized usingthe Bio-Rad multipurpose imager. The derived fractions of RNA unwound at the indicated time pointswere plotted against time (in minutes), and the data points were fit to a single-exponential equation(see Results) to determine the rates of unwinding (ku) of all substrates (Table 2). Error bars representSD from two separate experiments.

FIG 9 M-nsp13 does not unwind partially duplex RNA substrates with a 3= loading strand. Fourdifferent substrates with 3=-overhang lengths varying from 2 to 20 nucleotides were designed todetermine whether M-nsp13 can unwind in a 3=-to-5= direction. The helicase activity of M-nsp13(20 nM) was assessed on these substrates (5 nM each) at 30°C for 5 min in the presence of 2 mM ATP.The products were separated on a nondenaturing 8% polyacrylamide gel and visualized using theBio-Rad multipurpose imager.

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residual cellular ATP purified with the helicase as well as the longer overhang length of5=-RNA-20 substrate relative to other substrates that enhances M-nsp13 unwindingactivity.

Another important finding in this study is that the rate of unwinding of M-nsp13 isdirectly proportional to the length of the 5= loading strand of the partially duplex RNAsubstrate. This observation may be due to the ease of binding the long loading strandby M-nsp13.

Previous reports have shown that SARS-CoV nsp12 (RNA polymerase) and nsp13(helicase) physically interact, with nsp12 enhancing the activity of nsp13 (31, 37),thereby forming part of a larger SARS-CoV replication complex. The 5=-to-3= helicaseactivity of M-nsp13 may be similar to what was previously proposed for SARS-CoV (31).It was suggested that SARS-CoV nsp12 and nsp13 may work together to carry out oneof the replication complex functions, i.e., synthesis of subgenomic transcripts contain-ing the leader sequence derived from the 5= end of the genome (29, 30). To achievethis, nsp12 would have to transcribe RNA in a 3=-to-5= template direction, followed bypausing at transcription regulatory element sites. Such pauses may activate motion inthe opposite direction, allowing nsp13 (aided by nsp12 and possibly other viral andhost proteins) to unwind the 3= end of the nascent RNA primer and facilitate transferto the complementary region of the 5= leader genomic sequence.

Previous studies revealed that SARS-CoV and human coronavirus 229E helicasesrequired at least a 5-nt 5=-end single-strand overhang for efficient unwinding (31, 38).In this study, while up to 10 to 30% of ssRNA products were observed with the 0- to 2-nt5=-end single-strand overhang partially duplex RNA substrates, M-nsp13 exhibitedefficient unwinding activities only with partially duplex RNA substrates that have a�5-nt-long 5= loading strand.

While some helicases exhibit a clear preference for either RNA or DNA (9, 33–36),some helicases, such as SARS-CoV helicase, worked well with either RNA or DNA (31).Our findings with M-nsp13 also support the fact that coronavirus helicases do notexhibit a preference for either RNA or DNA substrates.

Comparison of the NTPase and dNTPase activities of M-nsp13 showed moderatevariations, as M-nsp13 clearly exhibited better enzymatic activities with the NTPs thanwith the dNTPs. Nonetheless, the data suggested that the nucleotide-binding site ofM-nsp13 has a low specificity among the NTPs. Similar data were also obtained forSARS-CoV nsp13 (30). Other RNA virus helicase-associated NTPase activities were alsoshown to lack any noticeable specificity for specific nucleotides (30, 35, 39–41). Thismay suggest that the lack of selectivity for specific nucleotide cofactors is a generalfeature of RNA virus helicases. Due to the high nucleotide consumption necessary formaximum viral RNA synthesis in the host cell, it may be beneficial for the helicase’sduplex-unwinding activity not to depend strictly on a specific nucleotide. As such, thereis lower risk for depletion of cellular NTP pools.

Other areas for future investigation include establishing the kinetic step size and thenatural state of the active protein, .i.e., the monomer of multimers, and characterizingreplication complex functions that involve M-nsp13, along with other MERS-CoV non-structural proteins.

TABLE 2 Kinetic parameters for M-nsp13 rates of unwinding and amplitudes for thedifferent partially duplex RNA substrates with 5= overhangs of various lengthsa

RNA substrate Amplitude ku (s�1)

5=-RNA-0 (0 ss, 22 ds) 0.27 � 0.04 0.07 � 0.035=-RNA-2 (2 ss, 22 ds) 0.40 � 0.06 0.08 � 0.035=-RNA-5 (5 ss, 22 ds) 0.63 � 0.03 0.17 � 0.035=-RNA-10 (10 ss, 22 ds) 0.90 � 0.02 0.42 � 0.025=-RNA-20 (20 ss, 22 ds) 0.92 � 0.02 1.29 � 0.14aThe rates of RNA unwinding (ku) and amplitudes were determined by fitting the data by means ofnonlinear regression to a single-exponent equation (see Results) using Prism 7.0 software. Experiments wereperformed two times.

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In conclusion, we report the first expression, purification, and biochemical charac-terization of MERS-CoV helicase. We showed that M-nsp13 required more ATP foroptimal unwinding of partially duplex RNA substrates with short 5= loading strands. Ourresults also revealed that the rate of unwinding of M-nsp13 increased with a partiallyduplex RNA substrate with a longer 5= loading strand. These results provide insightsthat enhance our understanding of the biochemical properties of M-nsp13. As such,M-nsp13 might be used as a target to identify inhibitors of MERS-CoV replication.

MATERIALS AND METHODSCloning, expression, and purification of M-nsp13 helicase. M-nsp13 is encoded by nucleotides 16208to 18001 of the MERS-CoV genome (human betacoronavirus strain 2c EMC/2012; GenBank accession no.JX869059.2). The coding region of M-nsp13 was amplified by PCR using 5=-BamHI-nsp13-CGGGATCCTGCTGTAGGCTCTTGTGTTG-3= as the forward primer and 3=-EagI-nsp13-ATGCGGCCGCTATTGCAGCTTGTAGTTGGTAAAGCTC-3= as the reverse primer. The PCR amplicons were digested with BamHI and EagI,followed by ligation into expression vector pET52b (Novagen-EMD Millipore), which fused M-nsp13 witha Strep tag at the N terminus. After confirmation of the amplicon nucleotide sequences, the constructwas transformed into E. coli BL21 cells. Protein expression and purification were performed as describedpreviously (1, 31, 42) using Strep-Tactin beads (IBA). The protein was eluted with 100 mM Tris, pH 8.0,150 mM NaCl, 10 mM desthiobiotin, 0.1% Triton X-100, 5 mM �-mercaptoethanol (BME), and 5% glycerol.Using dialysis, the buffer was exchanged with (100 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X-100,5 mM BME, and 5% glycerol). Fractions containing the desired protein were concentrated and stored at�80°C.

The M-nsp13_K288A mutant was prepared using the QuikChange II XL site-directed mutagenesis kit(Agilent) as described by the manufacturer and expressed and purified as described above for thewild-type enzyme.

Expression and purification of foot-and-mouth disease virus (FMDV) 3Dpol. Expression andpurification were as previously described (43). Briefly, the pET-28a-FMDV 3Dpol plasmid was transformedinto the Rosetta 2 expression strain (Novagen). Kanamycin-resistant colonies were grown at 37°C, andprotein expression was induced at an A600 of 0.9 to 1.0 by the addition of 1 mM isopropyl �-D-1-thiogalactopyranoside (IPTG). The cells were allowed to grow for 3 more h after induction. The cells wereharvested by centrifugation (4,500 � g, 20 min) and stored at �20°C. Frozen cell pellets were resus-pended in buffer A (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5% glycerol). The protein was purified bynickel affinity chromatography with a gradient of 25 mM to 500 mM imidazole in buffer A. Fractionscontaining pure protein (95%) were pooled and dialyzed against the storage buffer containing 12.5 mMTris-HCl, pH 8.0, 100 mM NaCl, and 50% glycerol.

Western blotting. Purified M-nsp13, M-nsp13K288A, and FMDV polymerase were separated by 10%SDS-polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membranes (Schlei-cher & Schuell, Dassel, Germany). M-nsp13 was detected with anti-M-nsp13 rabbit antiserum as theprimary antibody, which was commercially developed upon request (Abcam). A horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin G (Dako) was used as the secondary antibody.

Nucleic acid substrates. Synthetic oligonucleotides were purchased from Integrated DNA Technol-ogies (Coralville, IA). Sequences of the RNA and DNA substrates are shown in Fig. S1 in the supplementalmaterial. Concentrations were determined spectrophotometrically using absorption at 260 nm and theirextinction coefficients. Unlabeled oligonucleotides were annealed to corresponding 5=-Cy3-labeled22-mers in 50 mM Tris, pH 8.0, 50 mM NaCl at a ratio of 1 to 1.2 by heating them at 95°C for 5 min andcooling them slowly to room temperature. Unlabeled 22-mers were used as traps for the helicase assay.

Characterization of helicase activity. Helicase activity was measured by incubating 20 nM M-nsp13with a 5 nM concentration of an RNA substrate (Fig. S1) in a reaction buffer containing 20 mM HEPES,pH 7.5, 20 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mg/ml bovine serum albumin (BSA), 5 mM MgCl2, and2 mM ATP at 30°C for various times. Reactions were quenched by the addition of an equal volume ofloading buffer (0.2% SDS and 20% glycerol). Unless otherwise mentioned, reactant concentrations referto the final concentration in the reaction mixture. The released single-stranded RNA (ssRNA) product andunwound double-stranded RNA (dsRNA) were resolved by 8% nondenaturing PAGE using a runningbuffer containing 89 mM Tris-borate, pH 8.2, and run for 45 min at 4°C and 110 V. The controls formeasuring maximum unwinding were dsRNAs denatured by heat for 5 min at 95°C and loadedimmediately on the gel. In this and subsequent assays, the gels were scanned with a Bio-Rad multipur-pose imager. When necessary, the band intensities representing ssRNA and dsRNA were quantitatedusing the Image Lab 5.1 software (Bio-Rad). The fraction of unwound RNA was plotted against time, andthe kinetic parameters described in the mechanism were determined by nonlinear regression usingGraphPad Prism (GraphPad Inc.).

Analyses of RNA unwinding. To obtain the kinetic parameters associated with RNA unwinding byM-nsp13, the fraction of unwound RNA was plotted against time. By nonlinear regression, data were fitto the single-exponential equation in Results using Prism 7.0 (GraphPad Inc.).

To obtain the kinetic parameters associated with the Km and Vmax of M-nsp13 with regard to ATPhydrolysis, the fraction of unwound RNA was plotted against different ATP concentrations. Data fittingwas carried out by nonlinear regression to the Michaelis-Menten equation using Prism 7.0 (GraphPadInc.).

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SUPPLEMENTAL MATERIALSupplemental material for this article may be found at http://dx.doi.org/10.1128/mSphere.00235-16.

Figure S1, PDF file, 0.1 MB.

ACKNOWLEDGMENTThis work was supported by a Midwestern University new investigator start-up grant.

FUNDING INFORMATIONThis work, including the efforts of Adeyemi O. Adedeji, was funded by MidwesternUniversity New Investigator Start-Up Fund.

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